The measurement of various body constituents by the use of radioimmunoassay techniques has achieved widespread acceptance in recent years. Exemplary of substances which can be measured by radioimmunoassay using currently available commercial kits are ACTH (adrenocorticotropin), aldosterone, angiotensin I, angiotensin II, barbiturates, cyclic AMP, cyclic GMP, digoxin, folic acid, FSH (follicle stimulating hormone), gastrin, HB.sub.s Ag (hepatitis B antigen), HGH (human growth hormone), insulin, TSH (thyroid stimulating hormone), T4 (thyroxine), T3 (triiodothyronine), and vitamin B12.
Yalow and Berson, In Vitro Procedures with Radioisotopes In Medicine, International Atomic Energy Agency, Vienna (1970) pgs. 455 et seq., express the principle of radioimmunoassay in the following terms:
"Unlabelled antigen in unknown samples competes against labelled antigen ("tracer") for binding to antibody and thereby diminishes the binding of labelled antigen. The degree of competitive inhibition observed in unknown samples is compared with that obtained in known standard solutions for determination of concentration of antigen in unknowns." PA1 (i) Incubating a mixture of radioisotope-labelled antigen and unknown sample in a test tube which is coated with two layers of antibody; a first layer of nonspecific antibodies which is bound (chemically or physically) to the internal surface of the test tube, and a second layer of more specific antibodies which are bound (chemically or physically) to the nonspecific antibodies; PA1 (ii) Separating the radioisotope-labelled antigen (and with it, of course, the unlabelled antigen) bound to the specific antibodies from the free radioisotope-labelled antigen (and unlabelled antigen); PA1 (iii) Determining the radioactivity of the bound radioisotope-labelled antigen, the radioactivity of the free radioisotope-labelled antigen, or the radioactivity of each; PA1 (iv) Referencing the amount of bound radioisotope-labelled antigen, the amount of free radioisotope-labelled antigen or the ratio of bound/free or free/bound radioisotope-labelled antigen to corresponding values obtained by carrying out steps (i), (ii) and (iii) using as a sample known amounts of antigen; and PA1 (v) Determining the amount of antigen in the unknown sample using the relationship established in (iv). PA1 (a) Radioisotope-labelled antigen PA1 (b) Antigen standards of concentrations 0, 0.5, 1.0, 2.0, 3.0 and 5.0, nanograms of antigen per milliliter. PA1 (c) Antigen control serum of known concentration PA1 (d) Double antibody-coated polystyrene test tubes PA1 Add 50 .mu.l of 0 ng Standard to tubes 1 and 2. PA1 Add 50 .mu.l of 0.5 ng Standard to tubes 3 and 4. PA1 Add 50 .mu.l of 1.0 ng Standard to tubes 5 and 6. PA1 Add 50 .mu.l of 2.0 ng Standard to tubes 7 and 8. PA1 Add 50 .mu.l of 3.0 ng Standard to tubes 9 and 10. PA1 Add 50 .mu.l of 5.0 ng Standard to tubes 11 and 12. PA1 Add 50 .mu.l of Control Serum to tubes 13 and 14 and PA1 add 50 .mu.l of Test Sample to tubes 15 and 16. PA1 Add 1 ml of radioisotope-labelled antigen solution to all tubes. PA1 B=average CPM for a given set of tubes and PA1 B=average CPM for `0` ng tubes
The above-described type of radioimmunoassay procedure has come to be known as the "indirect" method of radioimmunoassay. Alternatively, the "direct" method of radioimmunoassay can be used to determine the presence or absence of a particular antigen in an unknown sample. In the "direct" method, labelled antibody is mixed with the unknown sample, which if it contains the antigen in question, will bind the labelled antibody.
In all radioimmunoassay procedures it is necessary to provide means for separating the bound from the free labelled tracer material. Many widely varied procedures have been developed and used; exemplary procedures are electrophoresis; chromatography; ion exchange; adsorption to dextran-coated charcoal, talc, or cellulose; and a number of solid-phase antibody techniques.
Two of the widely recognized solid-phase separation techniques comprise the covalent chemical bonding of an antibody to an insoluble polymeric substance or the physical adsorption of an antibody onto an insoluble polymeric substance; see, for example, Gurvich et al., Nature, 203:648 (1964); Wide et al., Biochim. Biophys. Acta., 130:257 (1966); Catt et al., Biochem. J., 100:31c (1966); Catt et al., J. Lab. Clin. Med., 70:820 (1967); Catt et al., Nature, 213:825 (1967); Axen et al., Nature, 214:1302 (1967); Catt et al., Science, 158:1570 (1967); Wide et al., Lancet, 2:1105 (1967); Salmon et al., J. Immunol., 103 (1):129 (1969); Catt, U.S. Pat. No. 3,646,346, issued Feb. 29, 1972; and Axen et al., U.S. Pat. No. 3,645,852, issued Feb. 29, 1972. The principal advantage of the solid-phase antibody separation techniques in radioimmunoassays is that they allow the isolation of bound from free labelled tracer material to be carried out by a relatively simple step at the completion of the immune reaction. This step may in practice, however, require several manipulations by the laboratory technician.
Catt's U.S. Pat. No. 3,646,346, issued Feb. 29, 1972, discloses an antibody-coated test tube as the means for carrying out a solid-phase separation. The tubes are prepared by adsorbing antibodies of the antigen to be tested on their internal surface. Adsorption is accomplished by incubating a buffered solution of the antibodies at room temperature for a period of several hours. Following the classical principles set down by Yalow and Berson, supra., a solution of labelled antigen and a solution of unlabelled antigen are incubated in the antibody-coated tubes causing the labelled and unlabelled antigens to compete for the available binding sites, and causing the formation of a two-phase system, i.e., a solid phase containing the bound antigen and a liquid phase containing the free antigen.
While the antibody-coated tube system has several advantages, it does have important drawbacks. Due to the unfavorable steric configuration of the antibody coated on the test tube, assay sensitivity is not as great as in some other systems. Furthermore, it is difficult, when preparing antibody-coated tubes, to maintain uniformity of the antibody coating from tube to tube.
Another solid-phase separation technique which has received recognition by the art is the double antibody solid-phase technique. The method is described by Parker in Radioimmunoassay of Biologically Active Compounds (1976), Prentice-Hall, Inc., Englewood Cliffs, New Jersey, pgs. 156-159 and by Hollander et al. in Radioimmunoassay Methods (1971), Churchill Livingston, Edinburgh, pgs. 419-422. The basic procedure comprises incubating a first antibody with labelled antigen and unlabelled antigen following the classical technique. Following the initial incubation a second antibody is added to the reaction system in an insolubilized form. This second antibody is of such a nature that it binds with the soluble antigen-antibody complex obtained in the first incubation to cause precipitation of the entire complex, i.e., antigen and double antibody. The second antibody is a nonspecific antibody and reacts with the antigen-antibody complex at sites on the more specific antibody which are non-reactive towards the antigen.
Parker, Radioimmunoassay of Biologically Active Compounds, supra., at pg. 157, states that "In some systems preformed first antibody-second antibody complexes bind effectively to antigen, permitting a more rapid assay, albeit with some diminution in sensitivity."
Bosma et al., J. Immunol., 115 (5):1381 (1975) deals with a solid phase radioimmunoassay that employs the "direct" method known as the sandwich technique. The technique of Bosma et al. comprises coating a plastic tube with a first coating of antigen followed by a second coating of divalent antibody. The antibody is chemically linked to the antigen with glutaraldehyde. The competitive reactions between labelled antigen and varying concentrations of unlabelled antigen are run in the coated tubes.
Immunochemical reactions have been used in tests for the determination of antigen or antibody other than radioimmunoassays; agglutination tests are exemplary. Schuurs' U.S. Pat. No. 3,551,555, issued Dec. 29, 1970, discloses the preparation of coated immunochemical reagent particles for use in agglutination reactions. According to the patent disclosure the reagent comprises finely divided solid carrier particles having adsorbed on the surface a protein inert to the determination method and next the antigen or antibody.